Extracellular matrix vesicles are associated with initial calcification in a variety of tissues. However, the mechanism by which they mediate mineralization is unclear. Cartilage matrix vesicles, produced by chondrocytes, are embedded in the extracellular matrix and are the initial site of apatite formation. Matrix vesicles contain metalloproteinases, including stromelysin-1 and 72 kDa gelatinase, which contribute to the degradation of matrix proteins. Breakdown of the matrix vesicles and release of their component enzymes follows apatite formation in these organelles, but relatively little is known concerning the mechanisms by which apatite crystals are deposited. Cartilage matrix vesicles are hypothesized to initiate apatite formation as follows: by providing a protected environment for accumulation of the calcium and phosphate ions necessary for mineralization; by presenting potential nucleators within the vesicle to serve as initiation sites for crystal formation, which is the focus of this proposal; by providing enzymes and growth factors that increase the influx of ions into the vesicles; and by providing enzymes that can alter the composition of the extracellular matrix. Previous studies indicate that proteolipids, together with calcium-phosphate-phospholipid complexes, are involved in the initiation of apatite formation. Our initial studies have shown that proteolipid can be isolated from matrix vesicles produced by rat costochondral cartilage cell cultures. Preliminary amino acid analysis of the isolated proteolipid suggests that we have isolated a novel peptide. The aims of this study are: 1) to determine the amino acid and cDNA sequences, covalently attached lipids and associated lipids of the Ca2+ and/or Pi-precipitating proteolipid component of califiable proteolipid isolated from matrix vesicles produced by rat chondrocyte cultures; 2) to generate and characterize polyclonal antibodies to purified matrix vesicle proteolipid; and 3) to test the efficacy of the tools generated in aims 1 and 2 by examining the regulation of proteolipid synthesis and incorporation into matrix vesicles by factors which regulate growth plate calcification.